Implantation and placental development are essential for the development of the mammalian embryo. The initial developmental decisions set aside three unique extraembryonic lineages--trophoblast, extraembryonic endoderm and extraembryonic mesoderm that form the early rudimentary placenta-like structures and, later, the definitive placenta. In the previous grant period we have concentrated on the growth and differentiation factors involved in the trophoblast and parietal endoderm (PE) lineages during peri-implantation mouse development. We identified a number of growth factors and receptors expressed by peri-implantation embryos and elucidated the functional significance of several of these for development. We discovered that parathyroid hormone-related peptide (PTHrP), a product of trophoblasts and decidua, regulates the differentiation program of PE cells through its modification of extracellular matrix and integrin specificity used for migration and differentiation. We produced a targeted mutation in the epidermal growth factor receptor (EGFr) and investigated its functional significance in growth and development. The morphogenetic effect of such molecules are mediated by transcriptional regulators. We have cloned a new basic helix loop helix (bHLH) transcription factor, Hxt, expressed in trophoblast and extraembryonic mesoderm, and found that it induces trophoblast-specific differentiation. In the present proposal we plan to investigate further the mechanisms influencing growth, cell division, differentiation and invasion during the peri-implantation period, and the role played in these events by maternal interactions. Specifically we will study the parameters and regulation of the cell cycle, cell proliferation and differentiation during early embryonic development, and trophoblast differentiation, and investigate the roles played by growth and differentiation factors in this process. We will then investigate the regulation of differentiation of extraembryonic cell lineages. Specifically we will investigate the mechanisms by which molecules such as PTHrP regulate differentiation and migratory and/or invasive behavior of PE and trophoblast in culture. Finally, we want to elucidate the interplay of maternal and embryonic interactions in the implanting embryo in vivo. Factors such as PTHrP and activin are expressed by maternal tissues and may regulate invasive and/or migratory behavior. We will concentrate on mechanisms regulating the invasive program with its temporal and spatial control of proteolytic activity to achieve definitive implantation in vivo. We will produce targeted mutations in molecules involved in the invasive program to define their specific functions in implantation. These experiments should lead to a better understanding of embryonic growth and differentiation around the time of implantation and the molecular nature of the cooperation between maternal and embryonic cells in achieving successful implantation and placenta formation. Because abnormal implantation and placenta formation are a major cause of embryo wastage and infertility, the experiments may provide a basis for improving the success of human pregnancy.